Aryl-alcohol Oxidase Involved in Lignin Degradation A MECHANISTIC STUDY BASED ON STEADY AND PRE-STEADY STATE KINETICS AND PRIMARY AND SOLVENT ISOTOPE EFFECTS WITH TWO ALCOHOL SUBSTRATES
Aryl-alcohol Oxidase Involved in Lignin Degradation A MECHANISTIC STUDY BASED ON STEADY AND PRE-STEADY STATE KINETICS AND PRIMARY AND SOLVENT ISOTOPE EFFECTS WITH TWO ALCOHOL SUBSTRATES
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DOI：
10.1074/jbc.m109.011593
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发表时间：
2009-09-11
影响因子：
4.8
通讯作者：
Medina, Milagros
中科院分区：
文献类型：
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作者：
Ferreira, Patricia;Hernandez-Ortega, Aitor;Medina, Milagros
芳基醇氧化酶（AAO）是GMC（葡萄糖-甲醇-胆碱氧化酶）家族中含有fad的酶。AAO参与木质素的真菌降解，这是一个高度生态和生物技术相关性的过程，通过提供木质素分解过氧化物酶所需的过氧化氢。在侧耳菇中，这种过氧化物是在对茴香醛的氧化还原循环中产生的，对茴香醛是一种细胞外真菌代谢物。除对茴香醇外，该酶还能氧化其他多不饱和伯醇。以对茴香醇和2,4-己二烯-1-醇为AAO模型底物，研究了其反应机理。稳态动力学参数和酶监测的周转符合O-2与还原AAO反应的顺序机制，然后释放醛产物。预稳态分析表明，AAO还原半反应在催化过程中是不可逆的和限速的。在稳定和准稳定状态条件下，底物和溶剂的动力学同位素效应（后者在使用双氘化底物时酶还原速度慢9倍，在同时评估底物和溶剂效应时酶还原速度慢13倍）揭示了一种同步机制，即氢化物从底物α -碳转移到FAD和质子从羟基提取同时发生。这与GMC氧化还原酶家族的其他成员提出的一般机制明显不同，后者意味着氢化物从先前稳定的底物醇盐转移。
Aryl-alcohol oxidase (AAO) is a FAD-containing enzyme in the GMC (glucose-methanol-choline oxidase) family of oxidoreductases. AAO participates in fungal degradation of lignin, a process of high ecological and biotechnological relevance, by providing the hydrogen peroxide required by ligninolytic peroxidases. In the Pleurotus species, this peroxide is generated in the redox cycling of p-anisaldehyde, an extracellular fungal metabolite. In addition to p-anisyl alcohol, the enzyme also oxidizes other polyunsaturated primary alcohols. Its reaction mechanism was investigated here using p-anisyl alcohol and 2,4-hexadien-1-ol as two AAO model substrates. Steady state kinetic parameters and enzyme-monitored turnover were consistent with a sequential mechanism in which O-2 reacts with reduced AAO before release of the aldehyde product. Pre-steady state analysis revealed that the AAO reductive half-reaction is essentially irreversible and rate limiting during catalysis. Substrate and solvent kinetic isotope effects under steady and presteady state conditions (the latter showing similar to 9-fold slower enzyme reduction when alpha-bideuterated substrates were used, and similar to 13-fold slower reduction when both substrate and solvent effects were simultaneously evaluated) revealed a synchronous mechanism in which hydride transfer from substrate alpha-carbon to FAD and proton abstraction from hydroxyl occur simultaneously. This significantly differs from the general mechanism proposed for other members of the GMC oxidoreductase family that implies hydride transfer from a previously stabilized substrate alkoxide.